Adaptive potential of maritime pine under contrasting environments.

BACKGROUND: Predicting the adaptability of forest tree populations under future climates requires a better knowledge of both the adaptive significance and evolvability of measurable key traits. Phenotypic plasticity, standing genetic variation and degree of phenotypic integration shape the actual and future population genetic structure, but empirical estimations in forest tree species are still extremely scarce. We analysed 11 maritime pine populations covering the distribution range of the species (119 families and 8 trees/family, ca. 1300 trees) in a common garden experiment planted at two sites with contrasting productivity. We used plant height as a surrogate of fitness and measured five traits (mean and plasticity of carbon isotope discrimination, specific leaf area, needle biomass, Phenology growth index) related to four different strategies (acquisitive economics, photosynthetic organ size, growth allocation and avoidance of water stress). RESULTS: Estimated values of additive genetic variation would allow adaptation of the populations to future environmental conditions. Overall phenotypic integration and selection gradients were higher at the high productivity site, while phenotypic integration within populations was higher at the low productivity site. Response to selection was related mainly to photosynthetic organ size and drought-avoidance mechanisms rather than to water use efficiency. Phenotypic plasticity of water use efficiency could be maladaptive, resulting from selection for height growth. CONCLUSIONS: Contrary to the expectations in a drought tolerant species, our study suggests that variation in traits related to photosynthetic organ size and acquisitive investment of resources drive phenotypic selection across and within maritime pine populations. Both genetic variation and evolvability of key adaptive traits were considerably high, including plasticity of water use efficiency. These characteristics would enable a relatively fast micro-evolution of populations in response to the ongoing climate changes. Moreover, differentiation among populations in the studied traits would increase under the expected more productive future Atlantic conditions.

Common microgeographical selection patterns revealed in four European conifers.

Microgeographical adaptation occurs when the effects of directional selection persist despite gene flow. Traits and genetic loci under selection can then show adaptive divergence, against the backdrop of little differentiation at other traits or loci. How common such events are and how strong the selection is that underlies them remain open questions. Here, we discovered and analysed microgeographical patterns of genomic divergence in four European and Mediterranean conifers with widely differing life-history traits and ecological requirements (Abies alba MIll., Cedrus atlantica [Endl.] Manetti, Pinus halepensis Mill. and Pinus pinaster Aiton) by screening pairs from geographically close forest stands sampled along steep ecological gradients. We inferred patterns of genomic divergence by applying a combination of divergence outlier detection methods, demographic modelling, Approximate Bayesian Computation inferences and genomic annotation to genomic data. Surprisingly for such small geographical scales, we showed that selection is strong in all species but generally affects different loci in each. A clear signature of selection was systematically detected on a fraction of the genome, of the order of 0.1%-1% of the loci depending on the species. The novel modelling method we designed for estimating selection coefficients showed that the microgeographical selection coefficient scaled by population size (Ns) was 2-30. Our results convincingly suggest that selection maintains within-population diversity at microgeographical scales in spatially heterogeneous environments. Such genetic diversity is likely to be a major reservoir of adaptive potential, helping populations to adapt under fluctuating environmental conditions.

Polygenic adaptation and negative selection across traits, years and environments in a long-lived plant species (Pinus pinaster Ait., Pinaceae).

A decade of genetic association studies in multiple organisms suggests that most complex traits are polygenic; that is, they have a genetic architecture determined by numerous loci, each with small effect-size. Thus, determining the degree of polygenicity and its variation across traits, environments and time is crucial to understand the genetic basis of phenotypic variation. We applied multilocus approaches to estimate the degree of polygenicity of fitness-related traits in a long-lived plant (Pinus pinaster Ait., maritime pine) and to analyse this variation across environments and years. We evaluated five categories of fitness-related traits (survival, height, phenology, functional, and biotic-stress response) in a clonal common-garden network planted in contrasted environments (over 20,500 trees). Most of the analysed traits showed evidence of local adaptation based on Qst -Fst comparisons. We further observed a remarkably stable degree of polygenicity, averaging 6% (range of 0%-27%), across traits, environments and years. We detected evidence of negative selection, which could explain, at least partially, the high degree of polygenicity. Because polygenic adaptation can occur rapidly, our results suggest that current predictions on the capacity of natural forest tree populations to adapt to new environments should be revised, especially in the current context of climate change.

Measuring recent effective gene flow among large populations in Pinus sylvestris: Local pollen shedding does not preclude substantial long-distance pollen immigration.

The estimation of recent gene flow rates among vast and often weakly genetically differentiated tree populations remains a great challenge. Yet, empirical information would help understanding the interaction between gene flow and local adaptation in present-day non-equilibrium forests. We investigate here recent gene flow rates between two large native Scots pine (Pinus sylvestris L.) populations in central Iberian Peninsula (Spain), which grow on contrasting edaphic conditions six kilometers apart from each other and show substantial quantitative trait divergence in common garden experiments. Using a sample of 1,200 adult and offspring chloroplast-microsatellite haplotypes and a Bayesian inference model, we estimated substantial male gametic gene flow rates (8 and 21%) between the two natural populations, and even greater estimated immigration rates (42 and 64%) from nearby plantations into the two natural populations. Our results suggest that local pollen shedding within large tree populations does not preclude long-distance pollen immigration from large external sources, supporting the role of gene flow as a homogenizing evolutionary force contributing to low molecular genetic differentiation among populations of widely distributed wind-pollinated species. Our results also indicate the high potential for reproductive connectivity in large fragmented populations of wind-pollinated trees, and draw attention to a potential scenario of adaptive genetic divergence in quantitative traits under high gene flow.

Evolutionary history of the mediterranean Pinus halepensis-brutia species complex using gene-resequencing and transcriptomic approaches.

KEY MESSAGE: Complementary gene-resequencing and transcriptomic approaches reveal contrasted evolutionary histories in a species complex. Pinus halepensis and Pinus brutia are closely related species that can intercross, but occupy different geographical ranges and bioclimates. To study the evolution of this species complex and to provide genomic resources for further research, we produce and analyze two new complementary sets of genetic resources: (i) a set of 172 re-sequenced genomic target loci analyzed in 45 individuals, and (ii) a set of 11 transcriptome assemblies. These two datasets provide insights congruent with previous studies: P. brutia displays high level of genetic diversity and no genetic sub-structure, while P. halepensis shows three main genetic clusters, the western Mediterranean and North African clusters displaying much lower genetic diversity than the eastern Mediterranean cluster, the latter cluster having similar genetic diversity to P. brutia. In addition, these datasets provide new insights on the timing of the species-complex history: the two species would have split at the end of the tertiary, and the changing climatic conditions of the Mediterranean region at the end of the Tertiary-beginning of the Quaternary, together with the distinct species tolerance to harsh climatic conditions would have resulted in different geographic distributions, demographic histories and genetic patterns of the two pines. The multiple glacial-interglacial cycles during the Quaternary would have led to the expansion of P. brutia in the Middle East, while P. halepensis would have been through bottlenecks. The last glaciations, from 0.6 Mya on, would have affected further the Western genetic pool of P. halepensis.

The GenTree Platform: growth traits and tree-level environmental data in 12 European forest tree species.

BACKGROUND: Progress in the field of evolutionary forest ecology has been hampered by the huge challenge of phenotyping trees across their ranges in their natural environments, and the limitation in high-resolution environmental information. FINDINGS: The GenTree Platform contains phenotypic and environmental data from 4,959 trees from 12 ecologically and economically important European forest tree species: Abies alba Mill. (silver fir), Betula pendula Roth. (silver birch), Fagus sylvatica L. (European beech), Picea abies (L.) H. Karst (Norway spruce), Pinus cembra L. (Swiss stone pine), Pinus halepensis Mill. (Aleppo pine), Pinus nigra Arnold (European black pine), Pinus pinaster Aiton (maritime pine), Pinus sylvestris L. (Scots pine), Populus nigra L. (European black poplar), Taxus baccata L. (English yew), and Quercus petraea (Matt.) Liebl. (sessile oak). Phenotypic (height, diameter at breast height, crown size, bark thickness, biomass, straightness, forking, branch angle, fructification), regeneration, environmental in situ measurements (soil depth, vegetation cover, competition indices), and environmental modeling data extracted by using bilinear interpolation accounting for surrounding conditions of each tree (precipitation, temperature, insolation, drought indices) were obtained from trees in 194 sites covering the species' geographic ranges and reflecting local environmental gradients. CONCLUSION: The GenTree Platform is a new resource for investigating ecological and evolutionary processes in forest trees. The coherent phenotyping and environmental characterization across 12 species in their European ranges allow for a wide range of analyses from forest ecologists, conservationists, and macro-ecologists. Also, the data here presented can be linked to the GenTree Dendroecological collection, the GenTree Leaf Trait collection, and the GenTree Genomic collection presented elsewhere, which together build the largest evolutionary forest ecology data collection available.

Bridging the genotype-phenotype gap for a Mediterranean pine by semi-automatic crown identification and multispectral imagery.

Progress in high-throughput phenotyping and genomics provides the potential to understand the genetic basis of plant functional differentiation. We developed a semi-automatic methodology based on unmanned aerial vehicle (UAV) imagery for deriving tree-level phenotypes followed by genome-wide association study (GWAS). An RGB-based point cloud was used for tree crown identification in a common garden of Pinus halepensis in Spain. Crowns were combined with multispectral and thermal orthomosaics to retrieve growth traits, vegetation indices and canopy temperature. Thereafter, GWAS was performed to analyse the association between phenotypes and genomic variation at 235 single nucleotide polymorphisms (SNPs). Growth traits were associated with 12 SNPs involved in cellulose and carbohydrate metabolism. Indices related to transpiration and leaf water content were associated with six SNPs involved in stomata dynamics. Indices related to leaf pigments and leaf area were associated with 11 SNPs involved in signalling and peroxisome metabolism. About 16-20% of trait variance was explained by combinations of several SNPs, indicating polygenic control of morpho-physiological traits. Despite a limited availability of markers and individuals, this study is provides a successful proof-of-concept for the combination of high-throughput UAV-based phenotyping with cost-effective genotyping to disentangle the genetic architecture of phenotypic variation in a widespread conifer.

Evolutionary rate and genetic load in an emblematic Mediterranean tree following an ancient and prolonged population collapse.

Severe bottlenecks significantly diminish the amount of genetic diversity and the speed at which it accumulates (i.e., evolutionary rate). They further compromise the efficiency of natural selection to eliminate deleterious variants, which may reach fixation in the surviving populations. Consequently, expanding and adapting to new environments may pose a significant challenge when strong bottlenecks result in genetic pauperization. Herein, we surveyed the patterns of nucleotide diversity, molecular adaptation and genetic load across 177 gene-loci in a circum-Mediterranean conifer (Pinus pinea L.) that represents one of the most extreme cases of genetic pauperization in widespread outbreeding taxa. We found very little genetic variation in both hypervariable nuclear microsatellites (SSRs) and gene-loci, which translated into genetic diversity estimates one order of magnitude lower than those previously reported for pines. Such values were consistent with a strong population decline that began some ~1 Ma. Comparisons with the related and parapatric maritime pine (Pinus pinaster Ait.) revealed reduced rates of adaptive evolution (α and ωa ) and a significant accumulation of genetic load. It is unlikely that these are the result from differences in mutation rate or linkage disequilibrium between the two species; instead they are the presumable outcome of contrasting demographic histories affecting both the speed at which these taxa accumulate genetic diversity, and the global efficacy of selection. Future studies, and programs for conservation and management, should thus start testing for the effects of genetic load on fitness, and integrating such effects into predictive models.

A multiscale approach to detect selection in nonmodel tree species: Widespread adaptation despite population decline in Taxus baccata L.

Detecting the molecular basis of local adaptation and identifying selective drivers is still challenging in nonmodel species. The use of purely population genetic approaches is limited by some characteristics of genetic systems, such as pleiotropy and polygenic control, and parallel evidence from phenotypic-based experimental comparisons is required. In long-lived organisms, the detection of selective pressures might also be precluded by evolutionary lag times in response to the environment. Here, we used the English yew to showcase an example of a multiscale integrative approach in a nonmodel species with limited plant and genomic resources. We combined information from two independent sources, phenotypes in a common environment and genomic data in natural populations, to investigate the signature of selection. Growth differences among populations in a common environment, and phenological patterns of both shoot elongation and male strobili maturation, were associated with climate clines, providing evidence for local adaptation and guiding us in the selection of populations for genomic analyses. We used information on over 25,000 SNPs from c. 1,200 genes to infer the demographic history and to test for molecular signatures of selection at different levels: SNP, gene, and biological pathway. Our results confirmed an overall demographic history of population decline, but we also found evidence for putative local adaptation at the molecular level. We identified or confirmed several candidate genes for positive and negative selection in forest trees, including the pseudo-response regulator 7 (PRR7), an essential component of the circadian clock in plants. In addition, we successfully tested an approach to detect polygenic adaptation in biological pathways, allowing us to identify the flavonoid biosynthesis pathway as a candidate stress-response pathway that deserves further attention in other plants. Finally, our study contributes to the emerging view that explaining contemporary standing genetic variation requires considering adaptation to past climates, especially for long-lived trees.

Looking for Local Adaptation: Convergent Microevolution in Aleppo Pine (Pinus halepensis).

Finding outlier loci underlying local adaptation is challenging and is best approached by suitable sampling design and rigorous method selection. In this study, we aimed to detect outlier loci (single nucleotide polymorphisms, SNPs) at the local scale by using Aleppo pine (Pinus halepensis), a drought resistant conifer that has colonized many habitats in the Mediterranean Basin, as the model species. We used a nested sampling approach that considered replicated altitudinal gradients for three contrasting sites. We genotyped samples at 294 SNPs located in genomic regions selected to maximize outlier detection. We then applied three different statistical methodologies-Two Bayesian outlier methods and one latent factor principal component method-To identify outlier loci. No SNP was an outlier for all three methods, while eight SNPs were detected by at least two methods and 17 were detected only by one method. From the intersection of outlier SNPs, only one presented an allelic frequency pattern associated with the elevational gradient across the three sites. In a context of multiple populations under similar selective pressures, our results underline the need for careful examination of outliers detected in genomic scans before considering them as candidates for convergent adaptation.

A Reference Genome Sequence for the European Silver Fir (Abies alba Mill.): A Community-Generated Genomic Resource.

Silver fir (Abies alba Mill.) is a keystone conifer of European montane forest ecosystems that has experienced large fluctuations in population size during during the Quaternary and, more recently, due to land-use change. To forecast the species' future distribution and survival, it is important to investigate the genetic basis of adaptation to environmental change, notably to extreme events. For this purpose, we here provide a first draft genome assembly and annotation of the silver fir genome, established through a community-based initiative. DNA obtained from haploid megagametophyte and diploid needle tissue was used to construct and sequence Illumina paired-end and mate-pair libraries, respectively, to high depth. The assembled A. alba genome sequence accounted for over 37 million scaffolds corresponding to 18.16 Gb, with a scaffold N50 of 14,051 bp. Despite the fragmented nature of the assembly, a total of 50,757 full-length genes were functionally annotated in the nuclear genome. The chloroplast genome was also assembled into a single scaffold (120,908 bp) that shows a high collinearity with both the A. koreana and A. sibirica complete chloroplast genomes. This first genome assembly of silver fir is an important genomic resource that is now publicly available in support of a new generation of research. By genome-enabling this important conifer, this resource will open the gate for new research and more precise genetic monitoring of European silver fir forests.

Inducibility of Plant Secondary Metabolites in the Stem Predicts Genetic Variation in Resistance Against a Key Insect Herbivore in Maritime Pine.

Resistance to herbivores and pathogens is considered a key plant trait with strong adaptive value in trees, usually involving high concentrations of a diverse array of plant secondary metabolites (PSM). Intraspecific genetic variation and plasticity of PSM are widely known. However, their ecology and evolution are unclear, and even the implication of PSM as traits that provide direct effective resistance against herbivores is currently questioned. We used control and methyl jasmonate (MJ) induced clonal copies of genotypes within families from ten populations of the main distribution range of maritime pine to exhaustively characterize the constitutive and induced profile and concentration of PSM in the stem phloem, and to measure insect herbivory damage as a proxy of resistance. Then, we explored whether genetic variation in resistance to herbivory may be predicted by the constitutive concentration of PSM, and the role of its inducibility to predict the increase in resistance once the plant is induced. We found large and structured genetic variation among populations but not between families within populations in resistance to herbivory. The MJ-induction treatment strongly increased resistance to the weevil in the species, and the genetic variation in the inducibility of resistance was significantly structured among populations, with greater inducibility in the Atlantic populations. Genetic variation in resistance was largely explained by the multivariate concentration and profile of PSM at the genotypic level, rather than by bivariate correlations with individual PSM, after accounting for genetic relatedness among genotypes. While the constitutive concentration of the PSM blend did not show a clear pattern of resistance to herbivory, specific changes in the chemical profile and the increase in concentration of the PSM blend after MJ induction were related to increased resistance. To date, this is the first example of a comprehensive and rigorous approach in which inducibility of PSM in trees and its implication in resistance was analyzed excluding spurious associations due to genetic relatedness, often overlooked in intraspecific studies. Here we provide evidences that multivariate analyses of PSM, rather than bivariate correlations, provide more realistic information about the potentially causal relationships between PSM and resistance to herbivory in pine trees.

Inferring selection in instances of long-range colonization: The Aleppo pine (Pinus halepensis) in the Mediterranean Basin.

Teasing apart the effects of natural selection and demography on current allele frequencies is challenging, due to both processes leaving a similar molecular footprint. In particular, when attempting to identify selection in species that have undergone a recent range expansion, the increase in genetic drift at the edges of range expansions ("allele surfing") can be a confounding factor. To address this potential issue, we first assess the long-range colonization history of the Aleppo pine across the Mediterranean Basin, using molecular markers. We then look for single nucleotide polymorphisms (SNPs) involved in local adaptation using: (a) environmental correlation methods (bayenv2), focusing on bioclimatic variables important for the species' adaptation (i.e., temperature, precipitation and water availability); and (b) FST -related methods (pcadapt). To assess the rate of false positives caused by the allele surfing effect, these results are compared with results from simulated SNP data that mimics the species' past range expansions and the effect of genetic drift, but with no selection. We find that the Aleppo pine shows a previously unsuspected complex genetic structure across its range, as well as evidence of selection acting on SNPs involved with the response to bioclimatic variables such as drought. This study uses an original approach to disentangle the confounding effects of drift and selection in range margin populations. It also contributes to the increased evidence that plant populations are able to adapt to new environments despite the expected accumulation of deleterious mutations that takes place during long-range colonizations.

De novo assembly of English yew (Taxus baccata) transcriptome and its applications for intra- and inter-specific analyses.

KEY MESSAGE: We provide novel genomic resources for Taxus baccata in the form of a reference transcriptome, SSR and SNP markers, and orthologous single-copy genes, useful for phylogenomic and population genomic applications. English yew (T. baccata) is the only European representative of the Taxaceae family, a conifer group originated in the Jurassic period. The wide extent of environmental heterogeneity within the species' range, together with its long presence in Europe, make English yew an ideal species to investigate adaptive evolution in conifers. To enlarge the genomic resources available for this species, we used Illumina short read sequencing followed by de novo assembly to build the transcriptome of English yew. In addition to a fully annotated transcriptome as well as large sets of new potential SSR and SNP markers for T. baccata, we provide a data set of orthologous single-copy genes across three Taxus species using Picea sitchensis as outgroup, and discuss ortholog uses and limitations for phylogenomic and population genomic applications.

High rate of adaptive evolution in two widespread European pines.

Comparing related organisms with differing ecological requirements and evolutionary histories can shed light on the mechanisms and drivers underlying genetic adaptation. Here, by examining a common set of hundreds of loci, we compare patterns of nucleotide diversity and molecular adaptation of two European conifers (Scots pine and maritime pine) living in contrasted environments and characterized by distinct population genetic structure (low and clinal in Scots pine, high and ecotypic in maritime pine) and demographic histories. We found higher nucleotide diversity in Scots pine than in maritime pine, whereas rates of new adaptive substitutions (ωa ), as estimated from the distribution of fitness effects, were similar across species and among the highest found in plants. Sample size and population genetic structure did not appear to have resulted in significant bias in estimates of ωa . Moreover, population contraction-expansion dynamics for each species did not affect differentially the rate of adaptive substitution in these two pines. Several methodological and biological factors may underlie the unusually high rate of adaptive evolution of Scots pine and maritime pine. By providing two new case studies with contrasting evolutionary histories, we contribute to disentangling the multiple factors potentially affecting adaptive evolution in natural plant populations.

Incorporating exposure to pitch canker disease to support management decisions of Pinus pinaster Ait. in the face of climate change.

Climate change is gravely affecting forest ecosystems, resulting in large distribution shifts as well as in increasing infection diseases and biological invasions. Accordingly, forest management requires an evaluation of exposure to climate change that should integrate both its abiotic and biotic components. Here we address the implications of climate change in an emerging disease by analysing both the host species (Pinus pinaster, Maritime pine) and the pathogen's (Fusarium circinatum, pitch canker) environmental suitability i.e. estimating the host's risk of habitat loss and the disease`s future environmental range. We constrained our study area to the Spanish Iberian Peninsula, where accurate climate and pitch canker occurrence databases were available. While P. pinaster is widely distributed across the study area, the disease has only been detected in its north-central and north-western edges. We fitted species distribution models for the current distribution of the conifer and the disease. Then, these models were projected into nine Global Climate Models and two different climatic scenarios which totalled to 18 different future climate predictions representative of 2050. Based on the level of agreement among them, we created future suitability maps for the pine and for the disease independently, which were then used to assess exposure of current populations of P. pinaster to abiotic and biotic effects of climate change. Almost the entire distribution of P. pinaster in the Spanish Iberian Peninsula will be subjected to abiotic exposure likely to be driven by the predicted increase in drought events in the future. Furthermore, we detected a reduction in exposure to pitch canker that will be concentrated along the north-western edge of the study area. Setting up breeding programs is recommended in highly exposed and productive populations, while silvicultural methods and monitoring should be applied in those less productive, but still exposed, populations.

Local effects drive heterozygosity-fitness correlations in an outcrossing long-lived tree.

Heterozygosity-fitness correlations (HFCs) have been used to understand the complex interactions between inbreeding, genetic diversity and evolution. Although frequently reported for decades, evidence for HFCs was often based on underpowered studies or inappropriate methods, and hence their underlying mechanisms are still under debate. Here, we used 6100 genome-wide single nucleotide polymorphisms (SNPs) to test for general and local effect HFCs in maritime pine (Pinus pinaster Ait.), an iconic Mediterranean forest tree. Survival was used as a fitness proxy, and HFCs were assessed at a four-site common garden under contrasting environmental conditions (total of 16 288 trees). We found no significant correlations between genome-wide heterozygosity and fitness at any location, despite variation in inbreeding explaining a substantial proportion of the total variance for survival. However, four SNPs (including two non-synonymous mutations) were involved in significant associations with survival, in particular in the common gardens with higher environmental stress, as shown by a novel heterozygosity-fitness association test at the species-wide level. Fitness effects of SNPs involved in significant HFCs were stable across maritime pine gene pools naturally growing in distinct environments. These results led us to dismiss the general effect hypothesis and suggested a significant role of heterozygosity in specific candidate genes for increasing fitness in maritime pine. Our study highlights the importance of considering the species evolutionary and demographic history and different spatial scales and testing environments when assessing and interpreting HFCs.

Molecular proxies for climate maladaptation in a long-lived tree (Pinus pinaster Aiton, Pinaceae).

Understanding adaptive genetic responses to climate change is a main challenge for preserving biological diversity. Successful predictive models for climate-driven range shifts of species depend on the integration of information on adaptation, including that derived from genomic studies. Long-lived forest trees can experience substantial environmental change across generations, which results in a much more prominent adaptation lag than in annual species. Here, we show that candidate-gene SNPs (single nucleotide polymorphisms) can be used as predictors of maladaptation to climate in maritime pine (Pinus pinaster Aiton), an outcrossing long-lived keystone tree. A set of 18 SNPs potentially associated with climate, 5 of them involving amino acid-changing variants, were retained after performing logistic regression, latent factor mixed models, and Bayesian analyses of SNP-climate correlations. These relationships identified temperature as an important adaptive driver in maritime pine and highlighted that selective forces are operating differentially in geographically discrete gene pools. The frequency of the locally advantageous alleles at these selected loci was strongly correlated with survival in a common garden under extreme (hot and dry) climate conditions, which suggests that candidate-gene SNPs can be used to forecast the likely destiny of natural forest ecosystems under climate change scenarios. Differential levels of forest decline are anticipated for distinct maritime pine gene pools. Geographically defined molecular proxies for climate adaptation will thus critically enhance the predictive power of range-shift models and help establish mitigation measures for long-lived keystone forest trees in the face of impending climate change.

Adaptive evolution of Mediterranean pines.

Mediterranean pines represent an extremely heterogeneous assembly. Although they have evolved under similar environmental conditions, they diversified long ago, ca. 10 Mya, and present distinct biogeographic and demographic histories. Therefore, it is of special interest to understand whether and to what extent they have developed specific strategies of adaptive evolution through time and space. To explore evolutionary patterns, the Mediterranean pines' phylogeny was first reconstructed analyzing a new set of 21 low-copy nuclear genes with multilocus Bayesian tree reconstruction methods. Secondly, a phylogenetic approach was used to search for footprints of natural selection and to examine the evolution of multiple phenotypic traits. We identified two genes (involved in pines' defense and stress responses) that have likely played a role in the adaptation of Mediterranean pines to their environment. Moreover, few life-history traits showed historical or evolutionary adaptive convergence in Mediterranean lineages, while patterns of character evolution revealed various evolutionary trade-offs linking growth-development, reproduction and fire-related traits. Assessing the evolutionary path of important life-history traits, as well as the genomic basis of adaptive variation is central to understanding the past evolutionary success of Mediterranean pines and their future response to environmental changes.

Using seedling and pericarp tissues to determine maternal parentage of dispersed valley oak recruits.

The spatial pattern of established seedlings yields valuable information about variation in fecundity, dispersal, and spatial structure of distributed recruits, but separating maternal and paternal contributions in monoecious species has been hampered by the "2 parent" problem. It is now possible to determine the maternal parentage of established recruits with genetic assay of maternally derived tissues of the seed or fruit, but the DNA of weathered maternal tissues often yields unreliable genotypes, reducing the practical range of such assay. We develop a mixed assay of seedling and seed (pericarp) tissues and illustrate it with distributed recruits of California valley oak (Quercus lobata Née). Detailed analysis indicates correct maternal assignment rates of canopy patch recruits of 56% (seedling assay only) versus 94% (mixed assay). For open patch recruits, maternal assignment rates were less than 50% (seedling assay only) versus 91% (mixed assay). The strategy of choice is to use seedling genotypes to identify a small set of credible parental candidates and then deploy 3-4 well-chosen pericarp/endocarp loci to reduce that list to a single obvious maternal candidate. The increase in the number of recruits available for subsequent analysis is pronounced, increasing precision and statistical power for subsequent inference.